Compositions prepared from poultry and methods of their use

ABSTRACT

Broth compositions prepared from poultry materials are disclosed. Selected poultry raw materials are processed to obtain a broth having high protein content and high chondroitin sulfate. Certain specific amino acids are present at higher concentration as compared to home-made broth and other commercial products. The disclosed broth compositions are effective in preventing and/or treating joint diseases and may also provide other nutritional and health benefits.

RELATED APPLICATIONS

This application claims priority to U.S. Patent application 61/843,662filed Jul. 8, 2013, the entire content of which is hereby incorporatedby reference into this application.

SEQUENCE LISTING

This application is accompanied by a sequence listing in a computerreadable form that accurately reproduces the sequences described herein.

BACKGROUND

Chicken broth is a complex mixture containing extractives obtained fromcooking of chicken parts or whole chickens. Different chicken broths mayhave different compositions which may explain the varying nutritionaland health values of different broths. One major constituent of achicken broth is soluble proteins, which are made up of albumins, alongwith many other proteins. Numerous other compounds are present inchicken broths. Examples of such compounds include, for example,minerals, organic compounds, nucleotides, metabolites, lipids,phospholipids, vitamins, among others.

Traditional home-style chicken broth is prepared by cooking one or morepoultry parts in water and/or steam for extended time. Broths preparedfrom different raw materials may have different compositions. Even whenthe same raw materials are used, slight variations of the cookingprocesses may result in broths having different constituent profiles.

SUMMARY

The present disclosure advances the art by providing broth compositionsprepared from poultry and methods of preparing and using the same. Inone embodiment, the disclosed composition may be prepared from poultry,such as, for example, chicken, turkey, or other birds. In anotherembodiment, the disclosed composition may be prepared using parts fromother animal sources. In another embodiment, the disclosed compositionmay be in the form of broth, stock, extract, or powder.

In one aspect, the composition prepared according to the disclosedmethods may have higher concentration of certain beneficial compounds.In another aspect, certain beneficial compounds may be enriched in thedisclosed compositions to a greater extent as compared to other brothproducts currently available on the market. In another aspect, one ormore ingredients may be supplemented in the broth to achieve certainhealth benefits that are not generally associated with chicken broth.Examples of such supplements may include but are not limited to ginger,coffee extract, ginseng, green tea, other botanicals such as willow barkor boswellia, curcumin/turmeric, omega-3 fatty acids, fish oil, krilloil, algal oil, Pycnogenol French maritime pine bark extract, grape seedextract, flax seed extract, ribonuclease, angiogenin, lactoferrin,ribonuclease-enriched lactoferrin, S-adenosyl methionine, collagen,collagen proteins or collagen peptides, gelatin, avocado/soybeanunsaponifiables (ASU), extract of hops cones, egg shell membrane,polypeptides derived from milk, such as casein or whey, MSM(Methylsulfonylmethane), Yucca, Devils claw, Bromelain, glutamic acid,cocoa, stinging nettle, Vitamin E, Vitamin D3, walnut extract, etc. Inone aspect, the disclosed broth may contain significant amount ofcollagen, collagen proteins and/or collagen peptides. In another aspect,significant amount of the collagen, collagen proteins and/or collagenpeptides may be present naturally in the disclosed broth. In anotheraspect, significant amount of the collagen, collagen proteins and/orcollagen peptides may be added into the disclosed broth as a supplement.

In home-style cooking, chicken or turkey broths may be made by cookingfor extended period of time in an open vessel or under high temperaturein a pressurized vessel. By contrast, this disclosure provides brothproducts having unique composition and methods of preparing the same.Also disclosed here are methods of selecting and preparing rawmaterials, cooking raw materials, and separating and purifying theresultant broths.

In one embodiment, raw materials from poultry or other animal sourcesmay be comminuted to a great extent to maximize extraction of variousbeneficial compounds. In one aspect, the raw materials may be reduced toa size of less than about 2 cm, 1 cm, 5 mm, 4 mm, 3 mm, 2 mm, or lessthan 2 mm. Mechanical processing of the poultry parts to small sizedparticles prior to cooking may help maximizing extraction of certaincompounds. In another embodiment, because the poultry parts have beenmechanically processed to very small-sized particles prior to cooking,more gentle cooking condition (e.g., at a temperature of 60-100° C.) andshorter cooking time (from about 8 minutes to 300 minutes) may be usedto obtain the broth from the processed poultry parts. Such gentler andshorter cooking may help prevent inactivation of certain compounds thatwould be otherwise inactivated if conventional processing and cookingmethods are used. Preservation of such compounds may explain thesuperior health benefits observed when the disclosed broths are testedagainst other broth products that are not prepared according to theinstantly disclosed methods.

In another embodiment, poultry parts with bones and cartilage may bemechanically separated and comminuted to fine pieces of less than 5 mm(millimeter), 4 mm, 3 mm, 2 mm, or 1 mm in size. In one aspect, no stepsare taken to remove the residual meat from the bones. These small piecesmay be cooked at about 70° C., 100° C., 110° C., 120° C., or as high as150° C., for at least 8 minutes, 15 minutes, 30 minutes, 1 hour, 2hours, 3 hours, or 6 hours or longer to maximize the extraction ofcertain broth fraction and/or compounds.

In another embodiment, the broth prepared according to the disclosedmethods show different protein profiles from those obtained from othercommercial broth products when analyzed by SDS-PAGE. In one aspect, atleast 10%, 20%, 30%, 40%, 50%, 70%, or 90% of the total proteins in thedisclosed compositions have a molecular weight of between 10 kD(kilo-Dalton) and 70 kD. In another aspect, at least 95% of the proteinsin the disclosed compositions have a molecular weight of less than 100kD. In another aspect, the methods may be used to further reducemolecular weight of the proteins in the broth to less than 10 kD, 5 kD,or even lower than 3 KD to enhance assimilation by a subject. By way ofexample, one or more enzymes that digest proteins may be used to reducethe molecular weight of the proteins in the broth.

In another embodiment, the disclosed composition may contain the aminoacids proline and histidine, wherein the ratio between proline andhistidine is at least 4:1 by weight. In one aspect, the proline ispresent in the composition by at least 8% (w/w), 10% or greater on solidbasis. In another aspect, the ratio between the proline and thehistidine is at least 6:1 by weight.

In another embodiment, the disclosed composition may contain the aminoacids glycine and histidine, wherein the ratio between the glycine andhistidine is at least 6:1 by weight. In one aspect, the glycine ispresent in the composition by at least 12%, 14% or greater (w/w) onsolid basis. In another aspect, the ratio between the glycine and thehistidine is at least 10:1 by weight.

In another embodiment, the disclosed composition may contain the aminoacids hydroxyproline and histidine, wherein the ratio between thehydroxyproline and histidine is at least 4:1 by weight. In one aspect,the hydroxyproline is present in the composition by at least 7%, 8% orgreater (w/w) on solid basis. In another aspect, the ratio between thehydroxyproline and the histidine is at least 6:1 by weight.

In another embodiment, the disclosed composition may contain proline,glycine, hydroxyproline and histidine. In one aspect, the ratio betweenthe glycine and the histidine is at least 6:1 by weight. In anotheraspect, the glycine is present in the composition by at least 12% (w/w)on solid basis. In another aspect, the ratio between the proline and thehistidine is at least 4:1 by weight. In another aspect, the ratiobetween the hydroxyproline and the histidine is at least 6:1 by weight.

In another embodiment, the disclosed composition may contain proline andother amino acids, and the amount of the proline is at least 10% byweight of the total amino acids in the composition. In anotherembodiment, the disclosed composition may contain glycine and otheramino acids, and the amount of the glycine is at least 20% by weight ofthe total amino acids in the composition. In another embodiment, thedisclosed composition may contain hydroxyproline and other amino acids,and the amount of the hydroxyproline is at least 8% by weight of thetotal amino acids in the composition.

In another embodiment, the disclosed composition may contain one or morebranched chain amino acids (BCAA) (e.g., valine, leucine andisoleucine), and the BCAAs in the composition are present at a higherlevel than the level of BCAAs in other broth products. In one aspect,the amount of total BCAAs, including valine, leucine and isoleucine, isat least 5%, 6%, or 7% by weight of total amino acids in thecomposition.

In another embodiment, the broth products prepared according to thisdisclosure may have a moisture protein ratio (MPR) of between 999:1 and1:999, or between 200:1 and 10:1. By way of example, a MPR of 200:1means the product contains 200 parts water and 1 part meat (protein). Inanother embodiment, the broth products prepared according to thisdisclosure may have a moisture protein ratio (MPR) of between 150:1 and40:1, or between 135:1 and 67:1.

Chondroitin sulfate (CS) is rich in cartilage and has been reported tobe beneficial for joint health. The selection of raw materials and theprocess by which the broth is prepared may contribute to the relativelyhigher levels of chondroitin sulfate in the disclosed composition. Inone embodiment of the present disclosure, the broth composition maycontain at least 6%, 7%, 8%, or 10% of chondroitin sulfate by weight oftotal dry solids in the composition, as measured by using enzymaticdigestion and LC-UV detection assay. See e.g., Ji et al., Journal ofAOAC International, Vol. 90, No. 3, 659-69 (2007), which is herebyincorporated by reference into this disclosure.

In one aspect, the composition may be in a liquid form ready to beconsumed or it may be in a concentrated liquid form such as a stock. Inanother aspect, the composition may be in a solid form, such as a powderor a paste.

In one embodiment, the composition may contain one or more polyphenols,wherein the concentration of the polyphenols is at least 4,000 μg/ml GAE(Gallic Acid Equivalent) based on Folin-Ciocalteu assay.

The broths disclosed herein may be characterized by the uniquecomposition as described above. The broths may also be characterized bythe methods through which they are prepared. Moreover, the disclosedbroths are also unique in the health benefits they may provide to asubject. Subjects may be divided into two groups, one group ingests (orconsumes) an effective amount of the disclosed broth product each day,while the other group ingests water, or other broth products. Thevarious indicators may then be measured and compared between the twogroups.

In one embodiment, the composition disclosed herein may reduce painsignificantly in a subject when it is administered to the subject ascompared to a subject not administered the composition. For purpose ofthis disclosure, “significantly” means the observed difference betweenthe treatment group and the control group is statistically significant.

Protein Kinase A (PKA) is a family of cyclic AMP (cAMP) dependentenzymes implicated in inflammation. In another embodiment, thecomposition may significantly reduce stress-induced expression ofprotein kinase A (PKA) in a subject administered said composition ascompared to a control subject not administered said composition. In oneaspect, the composition reduces expression of protein kinase A (PKA) by20%, 30%, by 50% or greater in a subject administered said compositionas compared to a control subject not administered said composition.

In another embodiment, the disclosed compositions may substantiallyinhibit activity of cyclooxygenase (COX)-2 as measured by using a COXInhibitor Screening Assay. In one aspect, the inhibition of COX-2 isgreater than 20%, 30%, or 40%, as compared with subject with noadministration of the composition. In another aspect, the disclosedcomposition causes no significant inhibition of COX1 as measured byusing a COX Inhibitor Screening Assay. In another aspect, the ratio ofinhibition between COX2 and COX1, i.e., COX2/COX1 exerted by the instantcomposition is between 2 and 30, between 5 and 20, or between 10 and 20.

In one embodiment, it is provided here methods for preventing ortreating various diseases by administering to a subject an effectiveamount of a composition prepared according to the instant disclosure. Inanother embodiment, the disclosed methods may further include a step ofidentifying subjects who are in need of treatments. In one aspect, thesubject may be susceptible to one or more of the diseases. In anotheraspect, the subject may already have one or more of the diseases.Examples of the diseases may include but are not limited to jointdisease, inflammation, autoimmune disease, diabetes, metabolic disorder,cognitive disorder and combination thereof.

In one aspect, the effective amount may be an amount of the compositionthat significantly reduces stress-induced expression of protein kinase A(PKA) as compared to a control subject who has not been administered thecomposition. In another aspect, the composition may reduce expression ofthe protein kinase A (PKA) by at least 50% in a subject administered thecomposition as compared to a control subject who has not beenadministered an effective amount of the composition.

In another aspect, the effective amount may be an amount of thecomposition that inhibits COX-2 activity by at least 20% in the subjectwhile not significantly inhibiting COX1 activity. In another aspect, theratio of inhibition between COX2 and COX1 exerted by the composition isbetween 2 and 30.

In another aspect, the effective amount is an amount of the compositionthat significantly reduces nociception (pain) as compared to a controlsubject who has not been administered an effective amount of thecomposition.

MicroRNAs (miRNAs) are small RNA molecules that can regulate geneexpression. miRNAs have been implicated in the development andprogression of many inflammatory diseases. In one embodiment, whenadministered to a subject, the composition may decrease expression of amiRNA in a subject administered an effective amount of said compositionas compared to a control subject not administered said composition.Examples of such miRNA may include but are not limited to SEQ ID Nos.1-16, 18-26, and 28-43.

In one embodiment, the disclosed methods help prevent and/or treatinflammation and/or autoimmune disease. In one aspect, the compositiondecreases expression of at least one miRNA in the subject receiving aneffective amount of the composition as compared to expression of thesame miRNA in a control subject not receiving the effective amount ofthe composition. Examples of the miRNA associated with these methodsinclude, for example, one or more of SEQ ID Nos. 1-16, and 18-26.

In another embodiment, the disclosed methods help prevent and/or treatdiabetes and/or a metabolic disorder. In one aspect, the compositiondecreases expression of at least one miRNA in the subject receiving aneffective amount of the composition as compared to expression of thesame miRNA in a control subject not receiving the effective amount ofthe composition. Examples of the miRNA associated with these methodsinclude, for example, one or more of SEQ ID Nos. 28-33.

In another embodiment, the disclosed methods help prevent and/or treat acognitive disorder. In one aspect, the composition decreases expressionof at least one miRNA in the subject receiving an effective amount ofthe composition as compared to expression of the same miRNA in a controlsubject not receiving the effective amount of the composition. Examplesof the miRNA associated with these methods include but are not limitedto one or more of SEQ ID Nos. 34-43. Examples of cognitive disordersinclude, for example, schizophrenia, autism, Alzheimer's disease, amongothers.

In another embodiment, when administered to a subject, the compositionmay increase expression of a miRNA in a subject administered aneffective amount of said composition as compared to a control subjectnot administered said composition. Examples of such miRNA may includebut are not limited to SEQ ID No. 17 and SEQ ID No. 27.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the protein profile by staining of proteins separated bySDS-PAGE.

FIG. 2 shows that up-regulation of PKA is significantly repressed inanimals fed with AAC1 broth as compared to animals fed with acommercially available product TSN.

FIG. 3 shows nocifensive responses of AAC1 compared to a commercialproduct.

FIG. 4 shows the protein profile by staining of proteins separated bySDS-PAGE and quantification of molecular weight distribution.

DETAILED DESCRIPTION

Chicken soup and chicken broth have been available for human consumptionfor centuries. Many studies have been performed that show various healthbenefits of chicken soup or broth. According to conventional home-stylemethod, chicken soup or broth is prepared by boiling whole chicken orlarge chicken parts in a pot of water for an extended period of time.The soup or broth prepared according to this method may not havemaximized the health promoting effects of the soup or broth because someof the compounds may have been lost during cooking or during processing,while other compounds may not have been extracted from the chickenparts.

The present disclosure provides an improved method by processing chickenparts prior to cooking and by controlling the processing temperature andcooking time to maximize the extraction of beneficial compounds while,concomitantly, minimizing loss of activity due to harsh processingconditions.

Specially selected raw materials from poultry are processed according tothe disclosed methods to obtain a broth having high protein content.Certain amino acids are present in relatively higher concentration ascompared to a home-made broth or other commercial products. As shown invarious animal studies described herein, the disclosed brothcompositions may prove effective in preventing and/or treating jointdiseases and the underlying pathology. The compositions may also provideother nutritional and health benefits such as decreasing inflammation.

The terms “broth” and “soup” refer to a liquid composition containing atleast one solute and may also be used to refer to a ready to serve form,a concentrate, a stock in either liquid or solid form.

The poultry broth of the disclosure may contain a significant amount ofdifferent amino acids. Such amino acids may be present in the form of aprotein or as free amino acids in the broth. Total amino acids includeboth amino acids present in the form of proteins and those present asfree amino acids. For purpose of this disclosure, the ratio of differentamino acids in a broth composition refers to the ratio between totalamino acids.

The term “dry solid” or “solid” as used herein refers to the componentsof a liquid composition that remain after all free liquid is removedfrom the liquid composition. In the case of an aqueous broth, the freeliquid is water.

The term “administer” means delivering of a material to an individual,such as by oral ingestion.

The term “subject” is used to refer to a mammal, including human being.

The term “substantially” means by at least 10-20%.

EXAMPLES

The following examples are provided for purposes of illustration of theembodiments only and are not intended to be limiting. The raw materials,reagents, chemicals, and other materials are presented as exemplarycomponents or reagents, and various modifications may be made in view ofthe foregoing discussion within the scope of this disclosure. Unlessotherwise specified in this disclosure, components, reagents, protocol,and other methods used in the system and the assays, as described in theExamples, are for the purpose of illustration only.

Example 1 Preparation of Broth from Turkey Parts

In this example, turkey parts were used to prepare a broth and theoverall quality and potential health benefits of the broth weredetermined. Briefly, raw turkey was mechanically separated and the partswere finely comminuted to less than 2 mm in size to maximize extraction.The small-sized parts from the turkey were gently cooked to 195° F. insteam for about 15 minutes or less. The broth was then separated fromthe insoluble fraction by decanting. Freed fat in the broth was alsoremoved from the broth by a centrifugal separator. The broth wasconcentrated in a commercial evaporator, chilled, and packaged for sale.

Example 2 Preparation of Broth from Chicken Parts

In this study, chopped chicken parts containing chicken bones andcartilage were cooked in water at a temperature greater than 250° F. formore than 6 hours to maximize the extraction of certain broth fractionand/or compounds. The broth obtained from this process was designated“AAC1” for internal reference. More particularly, USDA inspected choppedraw chicken bones remaining after major muscles were removed were cookedin water in a large commercial stainless steel cooking tank. Aftercooking, the liquid broth portion was separated from the chicken solidsby decanting. The broth was concentrated in a commercial evaporator thenspray dried and packed in labeled containers.

Example 3 Comparing the Protein and Amino Acid Profiles of DifferentBroths

Proteins are large molecules composed of one or more chains of aminoacids that perform a wide array of functions in biological systemsincluding, for example, functioning as enzymes, facilitating cellcommunication, and providing structural support to cells. Humans, aswell as other animals, obtain essential amino acids from proteinconsumed as part of their diet since they lack enzymes needed tosynthesize them. Ingestion of proteins leads to their break down intoamino acids through the digestive process. The amino acids can then beused in protein biosynthesis in muscle production and maintenance,glucose production, serve as a dietary nitrogen source, and serve as afuel source if necessary. The objective of this study was to determinethe concentration and size range of proteins in chicken broth AAC1 ascompared to a home-made product.

One sample prepared according to this disclosure (AAC1) and anothersample prepared according to home-style cooking methods (Homemade) werecompared. The percent solid (w/v) for AAC1 was 8% solid, while thepercentage (w/v) for Homemade was 1.7% solid.

Prior to determining the amount of protein by the Bradford method, eachsample was diluted in distilled water to a final 1% w/v solution. Astandard curve was prepared using bovine serum albumin (0-3.5 μg/μL).All samples were analyzed in triplicate. The amount of total protein wasdetermined using a plate reader at a wavelength of 595 nm. Results areshown in Table 1.

TABLE 1 Amount of total protein in broth samples Mean Sample ValuesResults Results Concentration SD CV AAC1 0.446 1.558 1.691 1.691 0.1327.8 0.461 1.693 0.474 1.821 Homemade 0.544 2.496 2.52 2.52 0.034 1.30.549 2.544

To correct for differences in the percent solids in AAC1 (8%) andHomemade (1.7%) samples, the protein values based on a 1% solution forAAC1 and Homemade were multiplied by their respective starting % solids.The final adjusted amount of total protein for AAC1 and Homemade areshown in Table 2.

TABLE 2 Adjusted total protein concentration in AAC1 and homemade brothAdjusted Sample Final Concentration % of AAC1 AAC1 13.6 μg/ul — Homemade 4.3 μg/ul 31.6%

To determine the protein profile of each sample, equal volumes of theAAC1 and Homemade samples (15.6 μl) were each mixed with Laemmli'ssample buffer and a reducing agent, heated at 95° C. for 5 minutes, andseparated on a 4-12% Bis Tris gel. The relative size range of theproteins was determined by comparison to a commercially availableprotein standard (ranging from 4.5 kDa to 300 kDa). A constant voltageof 150V was applied to the gel for 30 minutes, allowing for separationof proteins in each sample. The proteins were visualized in the gelusing SimplyBlue™ SafeStain.

The results are shown in FIG. 1. Lane 1 shows the protein profile forhomemade broth, lane 2 for the AAC1 broth, and lane 3 is molecularweight standard. Based on the protein profiles, AAC1 is composed ofapproximately 3 times more protein than a homemade product. Proteins inthe homemade product displayed a wider range of molecular weightdistribution, containing both large (up to about 300-500 kD) and smallproteins (about 5-15 kD). By contrast, the majority (i.e., greater than50%) of proteins in AAC1 has molecular weight of between 70 kDa and 15kDa.

Individual amino acid content was also analyzed. Table 3 below shows theindividual amino acid content of the broth compositions preparedaccording to the disclosed methods as compared to those prepared usinghome-style methods as well as other commercial products.

TABLE 3 Amino acid composition of different broths Table Two: Contentvalues above calculated to 100% Commercial Home- solids basis: 3823product Home- made- - W/W % % made-1 2 AAC1-1 AAC1-2 AAC1-3 TAURINE 2.50ASPARTIC ACID 3.63 2.44 3.63 3.47 7.04 5.34 5.38 THREONINE 1.44 0.761.52 1.42 1.75 2.13 1.79 SERINE 1.56 1.34 1.96 1.89 2.08 1.88 2.40GLUTAMIC ACID 10.25 6.98 9.44 9.26 10.52 9.50 10.14 PROLINE 3.53 3.055.93 5.57 9.59 10.66 11.07 HYDROXYPROLINE 2.19 2.99 5.31 4.92 na 7.009.39 GLYCINE 5.97 7.38 8.79 9.47 18.02 15.41 17.68 Lanthionine 0.06 0.000.00 0.00 0.00 0.00 0.00 ALANINE 3.91 3.37 4.60 4.58 8.06 7.53 7.53CYSTINE 0.44 0.29 0.34 0.40 0.00 0.25 0.17 VALINE 1.44 0.73 1.43 1.422.44 2.19 2.21 ISOLEUCINE 1.13 0.47 1.18 1.02 1.54 1.59 1.59 LEUCINE2.72 1.51 2.48 2.32 3.38 3.47 3.43 METHIONINE 0.81 0.52 0.93 0.77 1.011.06 1.10 TYROSINE 0.88 0.44 0.81 0.65 1.09 1.00 0.90 PHENYLALANINE 1.000.73 9.53 7.03 2.30 2.16 1.98 HYDROXYLYSINE 0.16 0.00 0.00 0.00 0.000.00 0.00 HISTIDINE 2.03 2.56 2.70 1.73 1.32 1.16 1.00 ORNITHINE 2.250.00 0.00 0.00 0.00 0.00 0.00 LYSINE 3.75 2.67 3.07 3.00 3.96 3.66 3.05ARGININE 3.06 2.79 4.01 3.75 8.24 6.69 6.61 TRYTOPHAN 0.16 0.06 0.120.09 0.22 0.15 Total 54.84 41.08 67.80 62.79 82.35 82.88 87.55

Example 4 Comparing Levels of Chondroitin Sulfate (CS) in DifferentBroths

Chondroitin sulfate (CS) is an important structural compound found incartilage and is implicated in joint health. CS has been shown to reducethe levels of many inflammatory mediators such as iNOS, PGE2, COX-2(Gwendolyn, Spine 2011), and NFkB (Vallières, Osteoarthritis Cartilage,2010). The goal of this study was to determine the amount of CS invarious chicken broth samples. Chicken broths were analyzed for total CSusing enzymatic digestion and LC-UV detection (Ji, Journal of AOACInternational, 2007). Results were calculated as area on the curvecompared to standard samples and the limit of quantification as 8 mgchondroitin sulfate/g dry material (Table 4). Among all chicken brothstested, AAC1 had the greatest amount of CS (8.797% w/w).

TABLE 4 Amounts of chondroitin sulfate (CS) Samples Average (mg) Drymg/g Dry % w/w Powdered Chicken 17.480 87.971 8.797 Broth AAC1 PowderedChicken 15.443 67.553 6.755 Broth H814 Frozen Concentrate 10.886 52.0595.206 TSN Frozen Concentrate 8.627 42.104 4.210 IDF Chicken 823 HomeStyle Broth 5.866 28.258 2.826 from Back Bones Frozen Concentrate 5.24326.227 2.623 Turkey 824 Powdered Chicken 4.979 24.335 2.434 Broth HML34511 Powdered Chicken 3.817 18.305 1.830 Broth A1004 Powdered Chicken3.075 15.054 1.505 Broth P1301 Home Style Broth 2.185 10.99 1.09 fromChicken Parts Home Style Broth 1.937 9.361 0.936 from Chicken Necks

Example 5 Comparison of Concentration of Polyphenols in DifferentChicken Broths

The concentration of polyphenols in different chicken broths wasdetermined using a modified Folin-Ciocalteau method (Slinkard, AmericanJournal of Enology and Viticulture, 1977). Polyphenols are a class ofchemical compounds known to have anti-oxidant and anti-inflammatoryproperties. While both AAC1 and home-style broths contained polyphenols,AAC1 contained 4828.8 μg/mL GAE of polyphenols, which is approximately 7times more GAE of polyphenols than a homemade broth (687.7 μg/mL GAE)made from the same kind of chicken parts.

Example 6 Effect of the Broth Composition on COX Family of Enzymes

The COX family of enzymes is responsible for the synthesis ofprostanoids such as prostaglandins. COX-1 is constitutively active whileCOX-2 is inducible and hence is upregulated during inflammation andpain. Compounds that block COX-2 activation while not effecting COX-1are of particular interest due to their ability to block inflammationwhile not causing unwanted side effects mediated by blocking COX-1activity. The level and specificity of COX inhibition by the chickenbroth prepared according to the disclosed methods was investigated.

Using an in vitro COX enzyme inhibitor assay (Abcam), chicken brothsamples were assayed for their ability to block COX-1 and COX-2 activityaccording to the manufacturer's protocol. The results are shown in Table5.

TABLE 5 Selective inhibition of COX-1 and COX-2 % Ratio of Sample EnzymeInhibition COX2/COX1Inhibition Powdered IDF AAC1 COX-1 −2.3 18.34 COX-242.4 Frozen IDF 823 COX-1 −51.5 0.43 COX-2 22.1 Home Style Broth fromCOX-1 −17.9 1.57 Back Bones COX-2 28.1 Home Style Broth from COX-1 −32.50.78 Necks COX-2 25.3 IDF Turkey 824 COX-1 −21.7 1.51 COX-2 32.7 ChickenBroth K COX-1 −25.5 1.21 COX-2 30.8 Commercial Broth COX-1 −5.1 5.16 TSNCOX-2 26.3 Commercial Broth COX-1 −39.9 0.28 H814 COX-2 11.3 CommercialBroth COX-1 −9.2 4.01 A1004 COX-2 36.7 Commercial Broth COX-1 −27.0 1.01HML COX-2 27.3 Commercial Broth COX-1 −19.9 2.36 PLNT COX-2 47.1 HomeStyle Broth from COX-1 −5.2 0.67 same raw parts COX-2 3.5

Broth labeled “Home Style Broth from same raw parts” was a brothprepared using traditional home-style cooking method with the same rawmaterials as those used for preparing AAC1. As shown in Table 5 above,chicken broth AAC1 showed the greatest ratio of COX-2 inhibition toCOX-1 inhibition (18.34) with greater than 40% inhibition of COX-2enzyme activity as compared to other commercial products or “Home StyleBroth from same raw parts.”

Example 7 Effect of the Disclosed Broth on Joint Diseases by RegulatingProtein Kinase A

Temporomandibular Joint Disorder (TMD) is a disease affecting thetemporomandibular or jaw joint (TMJ), the muscles of mastication, orboth. TMD is believed to be caused by activation of neurons and gliacells located in the trigeminal system (Tjakkes et al., 2010). Theprevalence of TMD symptoms have been reported in up to 93% in thegeneral population with varying incidence rates (Zhao et al., 2011).Further development of TMD may also lead to the development of achronically sensitized state of the disorder.

Protein kinase A (PKA) is a member of the family of cyclic AMP (cAMP)dependent enzymes that act as pro-inflammatory molecules in peripheraland central nervous systems. Increased levels of PKA expression bysensory nociceptive neurons has been reported during the chronicsensitized state. The objective of this study was to study the effectsof commercially available broths, home-style broths, and the brothsaccording to the instant disclosure. These various products may containcertain anti-inflammatory molecules that have some effects on TMD. Thesemolecules may exert their effects through PKA in regulating thedevelopment and progress of TMD.

Three chicken broths (AAC1, TSN, and a homemade broth) were investigatedin this study. Broth AAC1 (8% solids) was prepared at a concentration of0.5% (w/v). First, 5 g of powdered broth was placed in a clean,autoclaved bottle. The bottle was then filled to the 1 L mark withfiltered water and the mixture was stirred to allow the powder todissolve in water. A homemade style broth (1.7%) was also tested as acontrol. To achieve a dosage that would allow for the comparison of AAC1and the homemade-style broth, the homemade broth was diluted 16 foldwith filtered water by placing 62.5 mL of stock broth and diluting thebroth to 1 L. A commercially available broth, TSN, was also tested asanother control. To ensure TSN was tested at a concentration similar tothat of AAC1, the solution for TSN (33.3% solid) was made in a similarmanner. The TSN broth was allowed to warm until a consistency of thestock broth was non-gelatinous. Broths were measured out into 16.7 gincrements and placed into clean bottles as previously described. Thebottle was then filled to the 1 L mark with filtered water. Allsolutions were then placed in a warm, sonication bath for 30 minutes toallow all components of the broths to be evenly solubilized. Broths werestored in the refrigerator until they were administered to the animalsvia water bottle administration for 2 weeks prior to TMD induction.

Adult Sprague-Dawley male rats (200 g-300 g) were used in this study.The rats were divided into three groups, with each group being fed withwater, a commercial product (TSN), or AAC1 broth prepared according tothis disclosure.

Mechanical stress was applied to each group and upper spinal cordtissues containing the spinal trigeminal nucleus (STN) samples wereobtained and processed for immuno-staining. Prior to immunostaining,slides were placed at room temperature and covered with 1×PBS for 5minutes. PBS was removed, and liquid was replaced with a 5% normaldonkey serum—0.1% triton solution for 20 minutes. Tissues were thenwashed with 5 mL 1×PBS. Working dilutions of rabbit anti-rat PKA (BDBiosciences, San Jose, Calif.; 1:500) were made using 5% normal donkeyserum. Primary antibodies were allowed to incubate on tissues (100μl/tissue) for 3 hours at room temperature. Samples were then washedwith 5 mL 0.1% Tween 20-PBS and 2 mL 1×PBS. Working dilutions of donkeyanti-rabbit IgG Alexa 488® was made by diluting stock antibodies 1:200in 1×PBS. Secondary antibodies were allowed to incubate on sample slide(100 μl/tissue) for 1 hour at room temperature while protected fromlight. Slides were again washed with 5 mL 0.1% Tween 20-PBS and 2 mL1×PBS, and mounted for fluorescent analysis using Vectashieldfluorescent mounting media containing the fluorescent dye DAPI. Slideswere cover slipped, sealed using clear nail polish, and stored at 4° C.until microscopic images were collected.

A Zeiss Z1 imager with apotome was used to acquire 10× Z-Stacked imagesof the V3 areas of the trigeminal ganglia and medullary horn of the STN.Zen 2011 software was used to evenly balance the background of eachimage prior to analysis. Gray scale jpeg images were opened in ImageJsoftware, where 10 non-overlapping regions of interests (RIOs) with anarea of equal size were placed in areas representative of proteinexpression in each image, and the integrated pixel densities weremeasured. Background intensities were also acquired through similarprocedure, and averaged. The average background intensity acquired fromeach image was then subtracted from each integrated density values fromareas of interest. Subtracted integrated densities were averaged andfold changes were calculated as the average change±SEM from TMD controllevels. Statistical differences were determined using the Mann-Whitney Utest in SPSS software, and were considered to be different when p≤0.05.

The data in FIG. 2 provide evidence that elevated PKA levels caused byprolonged jaw opening are greatly repressed in AAC1 fed animals whencompared to levels in animals consuming either water or a commercialproduct (TSN). In Table 6, the results of several experiments aresummarized as the average fold change in the intensity of immunostainingwhen compared to water whose mean intensity was made equal to one.

TABLE 6 AAC1 repression of elevated PKA levels in response to jaw stresscaused by prolonged jaw opening Average P Value P Value Broth FoldChange (vs. TMD) (vs. TSN) Water (TMD) 1.00 ± 0.06 1.000 0.239 AAC1 0.75± 0.02 0.001 0.000 TSN 0.94 ± 0.04 0.239 1.000

The AAC1 broth disclosed herein showed a greater ability to modulate thelevels of PKA in the STN following joint stress when compared to acommercially available product. Based on the difference in compositionof AAC1 and a homemade broth, we would predict that AAC1 would besignificantly better at repressing stress-induced elevations in PKAlevels in the STN.

Example 8 Nocifensive Responses of AAC1 as Compared to Other BrothProducts

Temporomandibular Joint Disorder (TMD), is characterized by thecontinuation of pain behavior and sensation despite a decrease innociceptive inputs (Herb et al., 2006). The increased sensitivity can beattributed to the development of a chronically sensitized state of thenerves that provide sensory innervation of the joint, muscles,ligaments, and tendons. TMD patients often report that their symptomsnegatively affect other aspects of their life (Sessle, 2008). Some TMDpatients develop protective behavior modifications that may limit orminimize their everyday activities. This protective behavior is termed“nocifensive” behavior. The objective of this study was to determine ifAAC1 chicken broth could reduce TMJ stress-induced nocifensive behaviorsin response to mechanical stimulation, and how its performance comparesto homemade broth and commercially available products.

Three chicken broths (AAC1, TSN, and a home-style broth) wereinvestigated in this study. Broth AAC1 (8% solids) was made at a 0.5%(w/v) while a homemade style broth (1.7%) was tested at a dose thatwould allow for an equal comparison of the ratio of percent solidsbetween AAC1 and a homemade broth. Thus, the homemade broth was diluted8 fold with filtered water by placing 62.5 mL of stock broth, anddiluting the broth to 1 L. To test a competing commercially availablebroth at a similar concentration, a solution for TSN (33.3% solids) wasmade in a similar manner with the modification that the broth wasallowed to warm until a consistency of the stock broth wasnon-gelatinous.

Broths were measured out into 16.7 g increments and placed into cleanbottles as previously described. The bottle was then filled to the 1 Lmark with filtered water. All solutions were then placed in a warm,sonication bath for 30 minutes to allow all components of the broths toevenly solubilize. Broths were placed in the refrigerator until theywere administered to the animals via water bottle administration for 2weeks prior to TMD induction.

Adult Sprague-Dawley male rats (200 g-300 g) were housed separately inclean, standard plastic rat cages (VWR, West Chester, Pa.) withnon-restricted access to both food and water in a room with 12hour/light dark cycles. Three consecutive days prior to testing, animalswere allowed to enter the Ugo Basile Durham animal holding device (UgoBasile, Collegeville, Pa.) for 5 min to acclimate to testing conditions.During this acclimation period, stimulation of hair follicles andepidermis located in the masseter and TMJ region of the face occurred bygently rubbing the area with a pipette tip. This stimulation was used toimprove the condition of the animal to the testing procedure, thusreducing the number of false reactions to testing filaments. Baselinenocifensive behaviors were assessed by utilizing a modified version ofthe well-established von Frey method. A series of calibrated von Freyfilaments were applied in increasing force to the cutaneous area overthe masseter muscle. Prior to application, the muscle was palpated withthe tip of the testing filament to insure proper placement. Onceplacement was established, a scientist blinded to the experimentalconditions placed enough force on the location to accomplish a bend inthe filament. Reactions observed after initiation of force to the areaand prior to the bend of the filament, were verified by one otherscientist and recorded. Each filament was applied 5 times, and recordedas the number of reactions obtained from 5 applications of each specificcalibrated filament. Measurements were collected over both the right andleft masseter muscles of each animal, which were averaged together toobtain a combined average number of reactions out of 5. Baselinereadings were prior to 2 week feeding of all chicken broth products andagain 24 hours prior to TMD induction.

The number of nocifensive responses after 2 hr, 3 days, or 7 days inanimals experiencing a mechanically induced TMD pathology was measured.The results of this study, which are shown in FIG. 3, demonstrate thatAAC1 significantly reduces the number of nocifensive responses after 2hr, 3 days, or 7 days in animals experiencing a mechanically induced TMDpathology. AAC1 significantly decreases the number of nocifensiveresponses as compared to TMD animals that consumed only water. The TSNproduct did not cause a decrease in nocifensive responses at any of themeasured time points. Thus, while product AAC1 reduces nocifensivebehaviors in animals experiencing chronic inflammation resulting fromthe prolonged jaw opening TMD model, this ability is not exhibited by acommercially available product. Based on results obtained from previousexamples, as well as data presented in this example, we would predictthat a homemade broth product would not significantly reduce TMJ stressinduced nocifensive responses.

Example 9 Effect of Broth on Various Diseases Through Epigenetic Changes

The goal of this study was to evaluate the effects of dailyadministration of chicken broth on miRNAs implicated in the developmentand resolution of neurological inflammatory conditions and diseases.miRNAs are small RNA molecules (typically about 22-nucleotide long) thatplay an important role in regulating the genome of animals includinghumans. These molecules control the genetic expression of thousands ofgenes. Importantly, changes in the expression of miRNAs are implicatedin the development and progression of many inflammatory and diseasestates.

Adult Sprague-Dawley male rats (200 g-300 g) were housed separately inclean, standard plastic rat cages (VWR, West Chester, Pa.) withnon-restricted access to both food and water in a room with 12hour/light dark cycles. Chicken broth AAC1 (8% solids) and ahomemade-style broth (1.7% solids) were used in this test. Broth AAC1was made at a 1% (w/v) by placing 10 g (1%) of powdered broth in aclean, autoclaved bottle. The bottle was then filled to the 1 L markwith filtered water. To maintain comparison ratios of percent solidsbetween AAC1 and a homemade-style broth, the homemade broth was diluted8 fold with filtered water by placing 125 mL of stock broth, anddiluting the broth to 1 L. Solutions were then placed in a warm,sonication bath for 30 minutes to allow all components of the broths toevenly go into solution. Broths were placed in the refrigerator untilthey were to be used. Animals received 300 mL of broth every two daysfor a total of 4 weeks. Spinal trigeminal nucleus (STN) and frontalcortex (FC) samples were acquired from the animals, while pancreatictissue samples were obtained via abdominal dissection. Once extracted,tissues were immediately frozen in liquid nitrogen, and stored at −20°C. until RNA could be extracted.

Tissues were weighed, and placed into a 1.5 mL Eppendorf tube(Eppendorf, Hauppauge, N.Y.) containing 600 μl TRIzol® Reagent(Invitrogen, Grand Island, N.Y.). Tissues were homogenized using aplastic pestle in a clean, RNAase free hood. Once tissues werecompletely homogenized, 600 μl of TRIzol added to each sample, andinverted several times to ensure complete mixing. Once mixed, 120 μlchloroform was placed in each tube, and rapidly inverted. Samples werethen placed on ice for 5 minutes. After 5 minutes, samples werecentrifuged at 12,000×g for 15 minutes at 4° C. to separate the organicand aqueous layers. Once separated, the RNA containing aqueous layer wasremoved and placed into a clean, 1.5 mL tube. At this time, anadditional 120 μl chloroform was placed in each tube, and rapidlyinverted. Samples were again incubated on ice for 5 minutes andcentrifuged at 12,000×g for 15 minutes at 4° C. Once separated, the RNAcontaining aqueous layer was removed and placed into a clean, 1.5 mLtube. To precipitate the RNA, 1 mL of ice cold isopropanol was placed ineach tube. Each sample was then inverted several times to insurecomplete mixing. Samples were then incubated at −20° C. for 30 minutesand centrifuged at 12,000×g for 15 minutes at 4° C. RNA pellets werefirst visualized, and isopropanol was removed. RNA pellets were thenwashed with RNAase free 75% ethanol, and centrifuged 7,500×g for 10minutes at 4° C. Ethanol was then removed and the RNA pellets allowed todry. Pellets were then re-suspended in RNAase free water, and placed at−20° C.

cDNA was obtained from total RNA samples (250 ng) using the miScript 2RT Kit (Qiagen) according to manufacturer's manual. Samples wereincubated at 37° C. for 1 hour, followed by incubation at 95° C. for 5minutes. This cycle was completed once, at which time the sample wasplaced on ice. Samples not assayed immediately were placed at −20° C.for storage, while samples to be assayed immediately were diluted with200 μl of RNAase free water as described in manufacture's handbook.

Qiagen Pathway-Focused RT-PCR arrays were used to determine changes inmiRNA expression using panels focused on Diabetic, Inflammatory &Autoimmune, and Neurological Development & Diseases pathways. QuantiTextSYBR Green PCR Master Mix (2×), miScript Universal Primer (10×),template cDNA, and RNAase free water was allowed to come to roomtemperature prior to use. In a clean, RNAase-free environment thefollowing components were combined in the following amounts to a clean,RNAase-free reservoir as described in manufacturer's handbook. Oncemixed, 25 μl of prepared reaction was pipetted into each well of a 96well Pathway Focused RT-PCR plate using a multichannel pipette. Onceloaded, samples were covered with a provided, clear, plastic adhesivesheet, and each well sealed individually. Each plate was placed in anApplied Biosystems OneStep RT-PCR machine where it was incubatedinitially at 95° C. for 15 minutes, then 40 cycles at the followingconditions: 94° C. 15 seconds; 55° C. for 30 seconds; and 70° C. for 30seconds. Data collection occurred during the second step of each cycleof 40.

Once each experiment was completed, raw data was imported into OneStepsoftware. Prior to analysis, a baseline was established according tomanufacturer's specification listed in the protocol handbook (cycle 2—2cycles prior to amplification, not more than cycle 15). The baseline isthe noise level in early cycle, where there is no detectableamplification. At this point a threshold was also established accordingto manufacturer's specifications detailed in the handbook. Thresholdswere set using a logarithmic amplification plot to include thelog-linear range of the curve. The threshold was placed at 1 for allarrays. This position allowed the threshold to be set above backgroundnoise, and at the lower half of the linear portion of the curve for allcontrol samples. Under these conditions, raw CT values were obtained.

To obtain ΔCT values, raw CT values from one control sample (n=3) andone chicken broth sample (n=3) for each panel was placed in miScripmiRNA PCR Array Web-Based software provided by the manufacture. Data wasnormalized to a minimum of 2 housekeeping genes, since typically levelsof these genes do not change under experimental conditions. Onceappropriate housekeeping genes were selected, data was submitted, andnormalized ΔCT values were calculated by web-based software. Valuesobtained from data software were then placed into an excel spreadsheet.To calculate ΔΔCT values for chicken broth samples the followingequation was used: ΔΔCT_(AAC1)=ΔCT_(control sample1)−ΔCT_(AAC1 sample1).To determine fold change from control expression the following equationwas used: fold change=2{circumflex over ( )}ΔΔCT_(AAC1 sample1). Foldchanges obtained from 3 independent experiments were averaged, andreported as the average fold change±SDEV. Statistical significance wasdetermined through Mann-Whitney U, and significance determined whenp≤0.05.

TABLE 7 Changes in miRNA levels associated with inflammation and autoimmune diseases in response to AAC1 Average Fold Targets ChangeP Values If Control Vs. Name Pathology Known AAC1 Control Sequencerno-miR- Inflammation Fos, Ptgs2, 1.00 ± 0.07 0.05 UACAGUACUGU 101a-3pand Rac1, Socs2, 0.69 ± 0.07 GAUAACUGAA Autoimmune Spred1 SEQ ID No. 1rno-miR- Inflammation Fos, Ptgs2, 1.00 ± 0.06 0.046 UACAGUACUGU 101b-3pand Rac1, Socs2, 0.68 ± 0.05 GAUAGCUGAA Autoimmune Spred1 SEQ ID No. 2rno-miR- Inflammation F3, Il25, 1.00 ± 0.06 0.046 UAAAGUGCUGA 106b-5pand MgII, Osm. 0.65 ± 0.08 CAGUGCAGAU Autoimmune SEQ ID No. 3 rno-miR-Inflammation Il16, Irf4, 1.00 ± 0.05 0.046 UCCCUGAGACC 125b-5p andStat3, Tnfsf4.  0.78 ± 0.09 CUAACUUGUGA Autoimmune SEQ ID No. 4 rno-miR-Inflammation Bmp2, Fgf9, 1.00 ± 0.09 0.05 CAGUGGUUUUA 140-5p and Hdac7,0.78 ± 0.06 CCCUAUGGUAG Autoimmune Spred1, SEQ ID No. 5 Vegfa. rno-miR-Inflammation Ghr, Prlr, 1.00 ± 0.05 0.05 UGUAGUGUUUCC 142-3p and Rac1.0.69 ± 0.04 UACUUUAUGGA Autoimmune SEQ ID No. 6 rno-miR- InflammationBtla, Cd28, 1.00 ± 0.05 0.046 UAGCAGCACGU 16-5p and Fgf7, Ghr, 0.68 ±0.10 AAAUAUUGGCG Autoimmune Il10ra, SEQ ID No. 7 Spred1, Vegfa. rno-miR-Inflammation F3, Il25, 1.00 ± 0.10 0.05 CAAAGUGCUUAC 17-5p andMgII, Osm. 0.54 ± 0.08 AGUGCAGGUAG Autoimmune SEQ ID No. 8 rno-miR-Inflammation Btla, Cd28, 1.00 ± 0.06 0.05 UAGCAGCACAG 195-5p andFgf7, Ghr, 0.65 ± 0.12 AAAUAUUGGC Autoimmune Il10ra, SEQ ID No. 9Spred1, Vegfa rno-miR- Inflammation Bmp3, Cast, 1.00 ± 0.13 0.05UGUGCAAAUCUA 19a-3p and Cntfr, F3 0.45 ± 0.06 UGCAAAACUGA AutoimmuneSEQ ID No. 10 rno-miR- Inflammation Bmp3, Cast, 1.00 ± 0.09 0.05UGUGCAAAUCCA 19b-3p and Cntfr, F3 0.42 ± 0.04 UGCAAAACUGA AutoimmuneSEQ ID No. 11 rno-miR- Inflammation F3, Il25, 1.00 ± 0.09 0.046UAAAGUGCUUAU 20a-5p and MgII ,Osm. 0.62 ± 0.01 AGUGCAGGUAG AutoimmuneSEQ ID No. 12 rno-miR- Inflammation F3, Il25, 1.00 ± 0.06 0.05CAAAGUGCUCAU 20b-5p and MgII, Osm. 0.62 ± 0.05 AGUGCAGGUAG AutoimmuneSEQ ID No. 13 rno-miR- Inflammation CcI7, Cxcl12, 1.00 ± 0.15 0.05AUCACAUUGCC 23a-3p and Fas, Grem1, 0.77 ± 0.12 AGGGAUUUCC AutoimmuneIl11,Il3,Il6ra, SEQ ID No. 14 Kitlg, Mstn, Prkca, Prok2, Stat5b.rno-miR- Inflammation CcI7, Cxcl12,  1.00 ± 0.14 0.05 AUCACAUUGCC 23b-3pand Fas, Grem1, 0.74 ± 0.12 AGGGAUUACC Autoimmune Il11,Il3,Il6ra,SEQ ID No. 15 Kitlg, Mstn, Prkca, Prok2, Stat5b. rno-miR- InflammationBmp3, Cd28, 1.00 ± 0.10 0.05 UUCACAGUGGC 27a-3p and Csf1, Fgf1, 0.64 ±0.04 UAAGUUCCGC Autoimmune Grem1, Irf4, SEQ ID No. 16 Lifr, Mstn.rno-miR- Inflammation Bcl6, 1.00 ± 0.44 0.05 AAAGUGCUUCCA 291a-3p andCyp26b1, 8.13 ± 3.67 CUUUGUGUGCC Autoimmune Dock2, F3, SEQ ID No. 17Lefty1, Lefty2. rno-miR- Inflammation Hdac4, 1.00 ± 0.04 0.05UAGCACCAUUU 29c-3p and Il1rap, Lif,  0.88 ± 0.02 GAAAUCGGUUA AutoimmunePdgfa, Pdgfc, SEQ ID No. 18 Tnfrsf1a, Vegfa rno-miR- InflammationHdac5, Il1a, 1.00 ± 0.11 0.05 UGUAAACAUCC 30b-5p and Irf4, Lifr 0.64 ±0.16 UACACUCAGCU Autoimmune SEQ ID No. 19 rno-miR- InflammationHdac5, Il1a, 1.00 ± 0.10 0.05 UGUAAACAUCC 30d-5p and Irf4, Lifr 0.68 ±0.16 CCGACUGGAAG Autoimmune SEQ ID No. 20 rno-miR- InflammationHdac5, Il1a, 1.00 ± 0.14 0.05 UGUAAACAUCC 30e-5p and Irf4, Lifr 0.64 ±0.06 UUGACUGGAAG Autoimmune SEQ ID No. 21 rno-miR- InflammationBtla, Cd28, 1.00 ± 0.14 0.05 CAGCAGCAAUU 322-5p and Fgf7, Ghr, 0.63 ±0.14 CAUGUUUUGGA Autoimmune Il10ra, SEQ ID No. 22 Spred1, Vegfa rno-miR-Inflammation Areg, Bmp3, 1.00 ± 0.56 0.05 AGGCAGUGUAGU 34c-5p andIl6ra, Kitlg, 0.54 ± 0.14 UAGCUGAUUGC Autoimmune Nampt, SEQ ID No. 23Nfe2l1, Serpinf2 rno-miR- Inflammation Csf2, F3, 1.00 ± 0.10 0.05AAUAUAACACA 410-3p and Fgf7, Il4, 0.67 ± 0.05 GAUGGCCUGU AutoimmuneNr3c1, Vegfa SEQ ID No. 24 rno-miR- Inflammation Areg, Bmp3, 1.00 ± 0.060.046 UGGCAGUGUAU 449a-5p and Il6ra, Kitlg, 0.90 ± 0.01 UGUUAGCUGGUAutoimmune Nampt, SEQ ID No. 25 Nfe2l1, Serpinf2 rno-miR- InflammationF3, Il25, 1.00 ± 0.01 0.05 CAAAGUGCUGUU 93-5p and MgII, Osm 0.66 ± 0.04CGUGCAGGUAG Autoimmune SEQ ID No. 26

TABLE 8 Changes in miRNA levels associated with diabetes in response to AAC1 Average Fold Change Control P ValuesName Target Tissue AAC1 Vs. control Sequence rno-miR-Adipose, beta cells, 1.00 ± 0.02 0.05 UGGACGGAGAA 184 modulates insulin3.59 ± 2.86 CUGAUAAGGGU signaling, Down in SEQ ID No. 27 T2DM rno-miR-Adipose, Up in T1DM 1.00 ± 0.18 0.05 UUCAAGUAAUU 26b-5p 0.53 ± 0.10CAGGAUAGGU SEQ ID No. 28 rno-miR- Adipose, Increased 1.00 ± 0.33 0.05UAGCACCAUUUG 29b-3p then impaired glucose-  0.30 ± 0.12 AAAUCAGUGUUinsulin secreation SEQ ID No. 29 rno-miR- Adipose, Above 1.00 ± 0.550.05 UAGCACCAUUU 29c-3p 0.46 ± 0.15 GAAAUCGGUUA SEQ ID No. 30 rno-miR-Adipose, initiates 1.00 ± 0.07 0.05 UGUAAACAUCC 30a-5pglucotoxisity beta cell  0.82 ± 0.10 UCGACUGGAAG dysfunctionSEQ ID No. 31 rno-miR- Adipose, beta cells 1.00 ± 0.13 0.05 UGGCAGUGUCU34a-5p 0.42 ± 0.06 UAGCUGGUUGU SEQ ID No. 32 rno-miR- Adipose 1.00 ±0.21 0.05 GCCUGCUGGGG 370-3p 0.32 ± 0.10 UGGAACCUGGU SEQ ID No. 33

TABLE 9 Changes in miRNA levels associated with AAC1neurological development and in response to AAC1 Average Fold ChangeControl P Values Name Pathology AAC1 Vs. control Sequence rno-miR-  Autistic Disorders, 1.00 ± 0.10 0.05 UAAAGUGCUGACAGUGCAGAU 106b-5pSchizophrenia, 0.77 ± 0.13 SEQ ID No. 34 Alzheimer's rno-miR-Development, 1.00 ± 0.18 0.05 AGCUGGUGUUGUGAAUCAGGCCG 138-5pSchizophrenia 0.69 ± 0.20 SEQ ID No. 35 rno-miR-  Autistic Disorders1.00 ± 0.08 0.05 UCAGUGCAUCACAGAACUUUGU 148b-3p 0.74 ± 0.11SEQ ID No. 36 rno-miR- Schizophrenia 1.00 ± 0.05 0.046UAGCAGCACAGAAAUAUUGGC 195-5p 0.87 ± 0.04 SEQ ID No. 37 rno-miR-Alzheimer's, 1.00 ± 0.17 0.05 UGUGCAAAUCCAUGCAAAACUGA 19b-3pSpinocerebellar 0.65 ± 0.18 SEQ ID No. 38 Ataxia rno-miR- Schizophrenia1.00 ± 0.14 0.05 CAAAGUGCUCAUAGUGCAGGUAG 20b-5p 0.78 ± 0.12SEQ ID No. 39 rno-miR- Schizophrenia, 1.00 ± 0.06 0.05UUCAAGUAAUUCAGGAUAGGU 26b-5p Alzheimer's 0.76 ± 0.08 SEQ ID No. 40rno-miR- Prion Disease 1.00 ± 0.06 0.05 UCUCACACAGAAAUCGCACCCGU 342-3p0.81 ± 0.08 SEQ ID No. 41 rno-miR-  Schizophrenia 1.00 ± 0.08 0.05AUGUUGCUCGGUGAACCCC 409a-3p 0.72 ± 0.07 SEQ ID No. 42 rno-miR- Autistic Disorders 1.00 ± 0.12 0.05 UACGUCAUCGUCGUCAUCGUUA 598-3p 0.77 ±0.07 SEQ ID No. 43

Spinal cord samples obtained from animals fed 1% chicken broth AAC1 for28 days showed significant repression of 26 miRNAs implicated ininflammation as compared to the levels found in animals treated with thecontrol samples. Interestingly, miRNA of the 101 family (See Table 7),which has been shown to inhibit Map Kinase Phosphatase 1, were amongthose that are inhibited by AAC1 broth. Frontal cortex samples obtainedfrom animals fed 1% AAC1 chicken broth also showed a significantreduction in the expression of 10 miRNAs implicated in the developmentof multiple neurological diseases. These diseases include Autism,Schizophrenia, Alzheimer's, Spinocerebellar Ataxia, and Prion'sdiseases.

Taken together, the results from this study suggest that the chickenbroth AAC1 may be beneficial in the epigenetic prophylactic regulationof certain miRNAs implicated in neurological inflammation of the centralnervous system, the development and progression of certain neurologicaldiseases, as well as in the pathogenesis of metabolic syndrome. Thesedata suggest that chicken broth AAC1 is more effective in regulatingthese miRNAs than a homemade product prepared from the same rawmaterials.

Example 10 Protein Profiles and Quantification of Protein MolecularWeight Distribution

The goal of this study was to differentiate protein profiles indifferent chicken broth samples through gel electrophoresis. Gelelectrophoresis was performed on all the samples to separate proteincontent by molecular weight. Protein content was visualized using aSimple Blue Safe Stain. Approximate molecular weights of any discernableprotein bands were determined.

Two samples, a control sample containing most major proteins fromchickens (lane 2) and AAC1 broth (lane 3) were Diluted in DI water to afinal concentration of 1% Solid and analyzed by SDS-PAGE (FIG. 4) alongwith a molecular weight (MW) marker (lane 1). The distribution of majorprotein bands were quantitated. Different banding patterns may be aresult of different processing methods. As shown in FIG. 4, AAC1 had asmearing pattern indicating high levels of protein degradation which maybe attributed to the production process used to make this product. Atleast 90% of the proteins in AAC1 were within the range of 10-50 kD.This is in contrast with the control sample, which showed a much broaderdistribution of MW from 10 kD to 300 kD.

All animal studies described herein were performed using approvedprotocols in compliance with government rules and regulations. Changesmay be made in the above methods and systems without departing from thescope hereof. It should thus be noted that the matter contained in theabove description or shown in the accompanying drawings should beinterpreted as illustrative and not in a limiting sense. The followingclaims are intended to cover generic and specific features describedherein, as well as statements of the scope of the present method andsystem, which, as a matter of language, might be said to fall therebetween.

Although each of the embodiments described above has been illustratedwith various components having particular respective orientations, itshould be understood that the system and methods as described in thepresent disclosure may take on a variety of specific configurations withthe various components being located in a variety of positions andmutual orientations and still remain within the spirit and scope of thepresent disclosure. Furthermore, suitable equivalents may be used inplace of or in addition to the various components, the function and useof such substitute or additional components being held to be familiar tothose skilled in the art and are therefore regarded as falling withinthe scope of the present disclosure. Therefore, the present examples areto be considered as illustrative and not restrictive, and the presentdisclosure is not to be limited to the details given herein but may bemodified within the scope of the appended claims.

All references cited in this disclosure, including patents, patentapplications, scientific papers and other publications, are herebyincorporated by reference into this application.

We claim:
 1. A composition prepared from poultry parts, said compositioncomprising an enhanced quantity of chondroitin sulfate and havingcertain ratio between amino acids proline and histidine derived fromsaid poultry parts, wherein the amount of chondroitin sulfate derivedfrom said poultry parts is at least 6% by weight of total dry solids ofthe composition, and wherein said certain ratio between proline andhistidine derived from said poultry parts is at least 4:1 by weight,said composition being in solid or liquid form, wherein at least 50% ofproteins in said composition have a molecular weight of between 15 kDand 70 kD.
 2. The composition of claim 1, wherein said composition is ina liquid form and wherein said composition further comprises apolyphenol compound derived from said poultry parts at a concentrationof at least 4,000 μg/ml GAE (Gallic Acid Equivalent).
 3. The compositionof claim 1, wherein the ratio between proline and histidine derived fromsaid poultry parts is at least 6:1 by weight.
 4. The composition ofclaim 1, wherein said proline derived from said poultry parts is presentin said composition by at least 8% (w/w) on solid basis.
 5. Thecomposition of claim 1, further comprising glycine derived from saidpoultry parts, wherein the ratio between the glycine and histidinederived from said poultry parts is at least 6:1 by weight, and whereinsaid glycine is present in said composition by at least 12% (w/w) onsolid basis.
 6. The composition of claim 5, wherein the ratio betweenthe glycine and histidine derived from said poultry parts is at least10:1 by weight.
 7. The composition of claim 1, further comprisinghydroxyproline derived from said poultry parts, wherein the ratiobetween the hydroxyproline and histidine derived from said poultry partsis at least 4:1 by weight, and wherein said hydroxyproline derived fromsaid poultry parts is present in said composition by at least 7% (w/w)on solid basis.
 8. The composition of claim 7, wherein the ratio betweenthe hydroxyproline and histidine derived from said poultry parts is atleast 6:1 by weight.
 9. The composition of claim 1, further comprisingproline derived from said poultry parts, wherein the amount of theproline is at least 9% by weight of total amino acids.
 10. Thecomposition of claim 1, further comprising hydroxyproline, wherein theamount of the hydroxyproline derived from said poultry parts is at least7% by weight of total amino acids.
 11. The composition of claim 1, saidcomposition further comprising one or more branched chain amino acids(BCAA), said BCAA being at least one member selected from the groupconsisting of leucine, isoleucine, and valine, wherein the amount ofsaid BCAA derived from said poultry parts is at least 5% by weight oftotal amino acids in said composition.
 12. The composition of claim 11,wherein the amount of the BCAA derived from said poultry parts is atleast 6% by weight of total amino acids in said composition.
 13. Thecomposition of claim 1, further comprising at least one supplementedingredient selected from the group consisting of ginger, coffee extract,ginseng, green tea, willow bark, boswellia, curcumin/turmeric, omega-3fatty acids, fish oil, krill oil, algal oil, French maritime pine barkextract, grape seed extract, flax seed extract, ribonuclease,angiogenin, lactoferrin, ribonuclease-enriched lactoferrin, S-adenosylmethionine, collagen proteins, gelatin, avocado unsaponifiables, soybeanunsaponifiables, extract of hops cones, egg shell membrane, polypeptidesderived from milk, casein, whey, MSM (methylsulfonylmethane), yucca,devils claw, bromelain, glutamic acid, cocoa, stinging nettle, vitaminE, vitamin D3, walnut extract, and combination thereof.
 14. Thecomposition of claim 1, further comprising a collagen protein as asupplemented ingredient.
 15. The composition of claim 1, wherein saidcomposition has a moisture protein ratio (MPR) of between 999:1 and1:999.
 16. The composition of claim 1, wherein said poultry parts arederived from chicken.
 17. A method for preparing a composition frompoultry parts, comprising the steps of: (a) cutting whole bird or partsthereof to pieces having average size smaller than 2 cm, (b) cooking thepieces of step (a) at a temperature of between 70° C. and 150° C. forabout 8 to 360 minutes, and (c) removing insolubles to obtain thecomposition in a liquid form, wherein the amount of chondroitin sulfatein said composition is at least 7% by weight of total dry solids.